The strategy of this proposal is based on the rationale that identification of the inhibitor, substrate, proton translocation, and functionally relevant phosphorylation sites on monoamine transporters (VMAT2) will provide a basic understanding of the mechanism of action of monoamine sequestration into vesicles and the factors which regulate transporter activity. This work will be accomplished in three Specific Aims: (1) Identification of the reserpine binding site(s) on VMAT2. Novel reserpine photoaffinity labels will be synthesized and characterized, and photo-labelled peptides will be identified in order to map the reserpine binding site; (2) Identification of the substrate transport channel. This aim will involve the use of several approaches, including radioactive photo-activatable substrate analogs to covalently derivatize the substrate binding site on VMAT2; site-specific derivatization of VMAT2 at engineered cysteine residues with the cysteine-reactive reagents, methanethiosulfonate ethyl amine (MTSEA), and MTS-ethyltrimethylammonium (MTSET); and site-directed mutagenesis of potential residues lining the channel; (3) Determination of the functional role of two highly charged regions of VMAT2. This aim will involve the use of biochemical and genetic (site-directed mutagenesis) approaches to determine the role of phosphorylation of the N-terminus of VMAT2 on transporter function and the intracellular distribution/oligomeric state of the transporter. Reduced or aberrant activity of the monoamine transporter of the synaptic vesicles in dopaminergic neurons of the substantia nigra through either direct or indirect actions of toxicants (e.g., MPP+, insecticides) and genetically altered neuronally expressed proteins may play a central role in Parkinson's Disease. The regulation of uptake of monoamine neurotransmitters into storage vesicles may also play an important role in affective psychological disorders related to depression by altering levels of serotonin, norepinephrine, dopamine, or other neurotransmitters. This work will provide insight into the mechanism of action of the monoamine transporters and contribute to our understanding of how pharmacological and therapeutic strategies may be devised to treat Parkinsonism or other disorders of the nervous system.